Project Summary/Abstract Actin plays many vital roles in eukaryotic innate defense mechanisms against pathogenic microorganisms. Reciprocally, pathogens have developed various elegant and sophisticated ways to disrupt and/or usurp the actin cytoskeleton. By acting on the actin cytoskeleton, pathogenic toxins disturb cell morphology, cell motility, phagocytosis, epithelial permeability, and antigen presentation. Bacterial toxins not only represent targets for biomedical interventions, but having been tuned to the host cytoskeleton throughout millions of years of co- evolution, they foster our understanding of the cytoskeleton on molecular and cellular levels. The long-term goals are to learn pathogenic mechanisms employed by actin-specific toxins and to utilize the obtained knowledge to illuminate functions of the actin cytoskeleton in norm and pathology. One such poorly understood disruptive mechanism is implemented by the Actin Crosslinking Domain (ACD) toxin produced as a part of larger toxins by pathogenic strains of V.cholerae, V.vulnificus, A.hydrophila, and several other species of bacteria. ACD is an enzyme that covalently crosslinks monomeric actin into oligomers that cannot polymerize. The current paradigm of ACD pathogenesis suggests that the toxin merely depletes functional actin by causing accumulation of bulk amounts of polymerization-incompetent actin oligomers. Instead, this proposal suggests a radically different concept, according to which ACD initiates a unique toxicity cascade by converting actin monomers into highly toxic oligomers that potently disrupt actin dynamics when present at very low concentrations. The central hypothesis is that a unique combination of properties absent in G- and F-actin confers an exceptionally strong interaction of the oligomers with tandem organized G-actin binding proteins and enables them to potently disrupt several key steps of actin dynamics. Guided by strong preliminary data, this concept will be thoroughly tested by pursuing three specific aims: 1) Evaluate the effects of the ACD- crosslinked actin oligomers on actin dynamics controlled by mammalian formins, Arp2/3 complex, WH2 tandem nucleators, and Ena/VASP in solution and at a single filament level in vitro; 2) Confirm predicted cellular targets of the oligomers, identify novel targets, and study cellular effects of the oligomers using a combination of tandem affinity purification, immunoblotting, mass spectrometry, and functional assays; and 3) Apply the acquired knowledge for producing novel ACD-based and ACD-inspired tools for studying actin dynamics at the molecular and cellular levels. These goals will be achieved via a combination of biochemical, biophysical, cellular, analytical, and protein engineering approaches, all of which have been proven to be feasible in preliminary studies conducted by the applicant and his research team. The proposed study is both significant and innovative as it promises to fill a major gap in our understanding of pathogenic mechanisms employed by several life-threatening pathogens and permit the research team to utilize the acquired knowledge by creating tools for studying the role of tandem-organized actin regulators in actin dynamics.